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Education

Research summary

My PhD research focused on the design and validation of a combinatorial assay that allows the rapid and parallel screening of novel potential heterocycles that might be incorporated into DNA, from a library of heterocyclic amines for expansion of the genetic alphabet. I also explored the use of diamonds as a solid phase support for immobilizing DNA and studied their interaction with variety of enzymes.

Currently, I research on the development of molecular biology tools to be used in in vitro selection of functional DNA molecules, including those that bind to targets (aptamers) and catalyze reactions, as well as polymerases that manipulate unnatural DNA molecules. I am also actively working on the development of new design strategies to create libraries of oligonucleotides made of non-standard nucleoside analogs using isothermal amplification techniques to select for aptamers or DNA or RNA catalyst that perform like proteins.

Now, I am in the process of developing low-cost diagnostics systems to detect and differentiate infectious agents, including respiratory diseases (e.g. MERS-coV), noroviruses, mosquito-borne viruses (e.g. Zika, chikungunya and dengue viruses), drug-resistant HIV strains and tick-borne diseases. This work led to diagnostics platforms to detect viral diseases at or near points of sampling.

A list of recent publications are listed below. For a more complete list, please see my publications on the NCBI website or Google Scholar.

This paper combines two advances to detect MERS-CoV, the causative agent of Middle East Respiratory Syndrome, that have emerged over the past few years from the new field of "synthetic biology". Both are based on an older concept, where molecular beacons are used as the downstream detection of viral RNA in biological mixtures followed by reverse transcription PCR amplification. The first advance exploits the artificially expanded genetic information systems (AEGIS). AEGIS adds nucleotides to the four found in standard DNA and RNA (xNA); AEGIS nucleotides pair orthogonally to the A:T and G:C pairs. Placing AEGIS components in the stems of molecular beacons is shown to lower noise by preventing unwanted stem invasion by adventitious natural xNA. This should improve the signal-to-noise ratio of molecular beacons operating in complex biological mixtures. The second advance introduces a nicking enzyme that allows a single target molecule to activate more than one beacon, allowing "signal amplification". Combining these technologies in primers with components of a self-avoiding molecular recognition system (SAMRS), we detect 50 copies of MERS-CoV RNA in a multiplexed respiratory virus panel by generating fluorescence signal visible to human eye and/or camera.

Noroviruses are the major cause of global viral gastroenteritis with short incubation times and small inoculums required for infection. This creates a need for a rapid molecular test for norovirus for early diagnosis, in the hope of preventing the spread of the disease. Non-chemists generally use off-the shelf reagents and natural DNA to create such tests, suffering from background noise that comes from adventitious DNA and RNA (collectively xNA) that is abundant in real biological samples, especially feces, a common location for norovirus. Here, we create an assay that combines artificially expanded genetic information systems (AEGIS, which adds nucleotides to the four in standard xNA, pairing orthogonally to A:T and G:C) with loop-mediated isothermal amplification (LAMP) to amplify norovirus RNA at constant temperatures, without the power or instrument requirements of PCR cycling. This assay was then validated using feces contaminated with murine norovirus (MNV). Treating stool samples with ammonia extracts the MNV RNA, which is then amplified in an AEGIS-RT-LAMP where AEGIS segments are incorporated both into an internal LAMP primer and into a molecular beacon stem, the second lowering background signaling noise. This is coupled with RNase H nicking during sample amplification, allowing detection of as few as 10 copies of noroviral RNA in a stool sample, generating a fluorescent signal visible to human eye, all in a closed reaction vessel.

Because diamonds have strongly bonded networks of carbon atoms, they offer the potential to support DNA-targeted analysis in architectures that require very stable DNA immobilization with very low DNA leakage. Further, their non-porous structures should allow diamond-immobilized DNA to easily gain access to enzymes in bulk solution. As part of our work to develop a molecular biology tool kit to transform immobilized DNA, we asked whether diamond-immobilized DNA could be cleaved by sequence-specific restriction endonucleases, despite the large sizes of those enzymes, the potential for "steric" obstruction from the diamond surface, and the possibility that the diamond surface might inactivate those enzymes. We report here that both standard and "nicking" restriction endonucleases cut diamond-immobilized single-stranded DNA, after it forms a duplex with a complementary strand of DNA delivered from solution. As a somewhat surprising result, we also discovered that restriction enzymes could cleave a fraction of the immobilized duplex DNA even if the complementary strand came not from solution, but rather from a separate diamond crystallite. This cleavage did not result from a failure of the attachment linkage that allowed the diffusion of leaked DNA through bulk solvent. Rather, the cleavage required physical proximity between crystallites, as confirmed by transmission electron microscopy. These results add to the tools that can use diamond-immobilized DNA, as well as define practical constraints on assay architectures where diamond-immobilized DNA is presumed to be isolated from other diamond-immobilized DNA particles.

The preparation of a series of carbocyclic exo-amino nucleosides from selected primary aromatic amines in both the C1' α- and β-epimeric form as well as the corresponding building blocks for DNA synthesis is described. These nucleosides were incorporated into oligodeoxynucleotides and their base-pairing properties with natural bases and, in part, with themselves investigated. The results obtained confirm that such nucleoside analogues engage in specific interactions with natural nucleotides in DNA duplexes. In addition they can form self-pairs that match or exceed the stability of Watson-Crick base-pairs. Thus, carbocyclic exo-amino nucleosides can be taken into consideration in the design of novel base-pairs for the extension of the genetic alphabet, or for other applications in biotechnology.

A combinatorial assay was developed for screening a library of aromatic heterocyclic amines for their propensity to act as a complementary base Z in a DNA duplex. This assay may prove useful in speeding up the process of base-pair discovery for potential applications in biotechnology and synthetic biology.